Devices and processes for distribution of genetic material to mammalian limb

ABSTRACT

A process is described for the delivery of a therapeutic polynucleotide to limb muscle tissue suffering from or potentially suffering from ischemia. The polynucleotide is inserted into a mammalian limb vessel such as an artery. Delivery efficiency and distribution is enhanced by combining injection of a solution containing the polynucleotide with the use of an externally applied cuff.

FIELD

The invention relates to devices and processes for use in biologicalsystems. More particularly, processes that provide for the functionaldistribution of genetic material to mammalian cells are described.

BACKGROUND

Biotechnology includes the delivery of a genetic information to a cellto express an exogenous nucleotide sequence, to inhibit, eliminate,augment, or alter expression of an endogenous nucleotide sequence, or toexpress a specific physiological characteristic not naturally associatedwith the cell. Polynucleotides may be coded to express a whole orpartial protein, or may be anti-sense, or non-viral DNA, or recombinewith chromosomal DNA.

A basic challenge for biotechnology and thus its subpart, gene therapy,is to develop approaches for delivering genetic information to cells ofa patient in a way that is efficient and safe. This problem of “drugdelivery,” where the genetic material is a drug, is particularlychallenging. If genetic material are appropriately delivered they canpotentially enhance a patient's health and, in some instances, lead to acure. Therefore, a primary focus of gene therapy is based on strategiesfor delivering genetic material in the form of nucleic acids. Afterdelivery strategies are developed they may be sold commercially sincethey are then useful for developing drugs.

Delivery of a nucleic acid means to transfer a nucleic acid from acontainer outside a mammal to near or within the outer cell membrane ofa cell in the mammal. The term transfection is used herein, in general,as a substitute for the term delivery, or, more specifically, thetransfer of a nucleic acid from directly outside a cell membrane towithin the cell membrane. The transferred (or transfected) nucleic acidmay contain an expression cassette. If the nucleic acid is a primary RNAtranscript that is processed into messenger RNA, a ribosome translatesthe messenger RNA to produce a protein within the cytoplasm. If thenucleic acid is a DNA, it enters the nucleus where it is transcribedinto a messenger RNA that is transported into the cytoplasm where it istranslated into a protein. Therefore if a nucleic acid expresses itscognate protein, then it must have entered a cell. A protein maysubsequently be degraded into peptides, which may be presented to theimmune system.

It was first observed that the in vivo injection of plasmid DNA intomuscle enabled the expression of foreign genes in the muscle (Wolff, J.A., Malone, R. W., Williams, P, et al. Direct gene transfer into mousemuscle in vivo. Science 1990;247:1465–1468.). Since that report, severalother studies have reported the ability for foreign gene expressionfollowing the direct injection of DNA into the parenchyma of othertissues. Naked DNA was expressed following its injection into cardiacmuscle (Acsadi, G., Jiao, S., Jani, A., Duke, D., Williams, P., Chong,W., Wolff, J. A. Direct gene transfer and expression into rat heart invivo. The New Biologist 3(1), 71–81, 1991.).

SUMMARY

In a preferred embodiment, a process is described for delivering apolynucleotide into a parenchymal cell of a mammal, comprising making apolynucleotide such as a nucleic acid, inserting the polynucleotide intoa mammalian vessel, such as a blood vessel, increasing the permeabilityof the vessel, and delivering the polynucleotide to the parenchymal cellthereby altering endogenous properties of the cell. Increasing thepermeability of the vessel consists of increasing pressure againstvessel walls and/or inhibiting the flow of fluid through the vessel.

In another preferred embodiment, an in vivo process for delivering apolynucleotide to a parenchymal cell of a mammal is described. First,the polynucleotide is inserted into a blood vessel. Interior blood flowis then externally impeded and the polynucleotide is delivered to theparenchymal cell. The polynucleotide may consist of naked DNA, a viralparticle/vector, a non-viral vector or may be a blocking polynucleotidefor preventing gene expression. The parenchymal cell may consist of amuscle cell, such as a limb (leg or arm) muscle cell.

The process includes externally impeding interior blood flow byexternally applying pressure to interior blood vessels such ascompressing mammalian skin by applying a tourniquet over the skin.Compressing mammalian skin also includes applying a cuff over the skinsuch as a sphygmomanometer.

In another preferred embodiment, an in vivo process for delivering apolynucleotide to a mammalian cell consists of inserting thepolynucleotide into a blood vessel and applying pressure to one or moreblood vessels. The pressure is applied externally to mammalian skin andthe polynucleotide is delivered to the mammalian cell. However, it isimportant that the function of the mammal's limbs is not permanentlyimpaired using this process. The process especially consists of apolynucleotide delivered to non-vascular (not of the smooth muscle cellssurrounding a vessel) parenchymal cells.

In yet another preferred embodiment, a device for applying pressure tomammalian skin for in vivo delivery of a polynucleotide to a mammaliancell is described. The device consists of a cuff, as defined in thisspecification, applied to mammalian skin to impede fluid flow throughthe vessel thereby increasing delivery efficiency of the polynucleotideto the mammalian cell.

In a preferred embodiment it may be preferential to immunosuppress thehost receiving the nucleic acid. Immunosuppression can be long term orfor a short duration, preferably around the time of nucleic aciddelivery. This can be accomplished by treatment with (combinations of)immunosuppresive drugs like cyclosporin A, ProGraf (FK506),corticosteroids, deoxyspergualin, and dexamethason. Other methodsinclude blocking of immune cell activation pathways, for instance bytreatment with (or expression of) an antibody directed against CTLA4;redirection of activated immune cells by treatment with (ore expressionof) chemokines such as MIP-1a, MCP-1 and RANTES; and treatment withimmunotoxins, such as a conjugate between anti-CD3 antibody anddiphtheria toxin.

In a preferred embodiment, the process may be used to deliver atherapeutic polynucleotide to a muscle cell for the treatment ofvascular disease or occlusion. The delivered polynucleotide can expressa protein or peptide that stimulates angiogenesis, vasculogenesis,arteriogenesis, or anastomoses to improve blood flow to a tissue. Thegene may be selected from the list comprising: VEGF, VEGF II, VEGF-B,VEGF-C, VEGF-D, VEGF-E, VEGF₁₂₁, VEGF₁₃₈, VEGF₁₄₅, VEGF₁₆₅, VEGF₁₈₉,VEGF₂₀₆, hypoxia inducible factor 1α (HIF 1α), endothelial NO synthase(eNOS), iNOS, VEFGR-1 (Flt1), VEGFR-2 (KDR/Flk1), VEGFR-3 (Flt4),neuropilin-1, ICAM-1, factors (chemokines and cytokines) that stimulatesmooth muscle cell, monocyte, or leukocyte migration, anti-apoptoticpeptides and proteins, fibroblast growth factors (FGF), FGF-1, FGF-1b,FGF-1c, FGF-2, FGF-2b, FGF-2c, FGF-3, FGF-3b, FGF-3c, FGF-4, FGF-5,FGF-7, FGF-9, acidic FGF, basic FGF, hepatocyte growth factor (HGF),angiopoietin 1 (Ang-1), angiopoietin 2 (Ang-2), Platelet derived growthfactors (PDFGs), PDGF-BB, monocyte chemotactic protein-1, granulocytemacrophage-colony stimulating factor, insulin-like growth factor-1(IGF-1), IGF-2, early growth response factor-1 (EGR-1), ETS-1, humantissue kallikrein (HK), matrix metalloproteinase, chymase,urokinase-type plasminogen activator and heparinase. The protein orpeptide may be secreted or stay within the cell. For proteins andpeptides that are secreted, the gene may contain a sequence that codesfor a signal peptide. The delivered polynucleotide can also suppress orinhibit expression of an endogeneous gene or gene product that inhibitsangiogenesis, vasculogenesis, arteriogenesis or anastomosis formation.Multiple polynucleotides or polynucleotides containing more that onetherapeutic gene may be delivered using the described process. The geneor genes can be delivered to stimulate vessel development, stimulatecollateral vessel development, promote peripheral vascular development,improve blood flow in a muscle tissue, or to improve abnormal cardiacfunction. The gene or genes can also be delivered to treat peripheralcirculatory disorders, myocardial disease, myocardial ischemia, limbischemia, arterial occlusive disease, peripheral arterial occlusivedisease, vascular insufficiency, vasculopathy, arteriosclerosisobliterans, thromboangiitis obliterans, atherosclerosis, aortitissyndrome, Behcet's disease, collagenosis, ischemia associated withdiabetes, claudication, intermittent claudication, Raynaud disease,cardiomyopathy or cardiac hypertrophy. The polynucleotide can bedelivered to a muscle cell that is suffering from ischemia or a normalmuscle cell. The muscle cell can be a cardiac cell or a skeletal musclecell. A preferred skeletal muscle cell is a limb skeletal muscle cell.The polynucleotides can also be delivered to a cells in a tissue that isat risk of suffering from ischemia or a vascular disease or disorder.

Further objects, features, and advantages of the invention will beapparent from the following detailed description when taken inconjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A & 1B. Photomicrographs of muscle sections histochemicallystained for β-galactosidase expression. Panel A represents a muscle(pronator teres) with a high level of expression; panel B represents amuscle (abductor pollicis longus) with an average level of expression.Magnification: 160×.

FIG. 2A–2C. Expression of β-galactosidase (light grey) and GFP (white)in rat muscle injected intraarterially at different times with therespective expression pDNAs. Panel A (640× magnification) is a low-powerfield illustrating that expression of β-galactosidase and GFP weretypically not co-localized. Panels B and C are high power fields (1600×magnification) that show an example of co-localization (B) and separateexpression (C).

FIG. 3A–3C. Muscle sections obtained 5 min (A and B) and 1 h (C) after50 μg of Rh-pDNA in 10 ml of normal saline were injected within 7 secinto the femoral artery of rat without impeding the outflow (A) orimpeding outflow (B and C). Arrows indicate Rh-pDNA between cells andarrowheads indicate pDNA inside myofibers. Magnification: 1260 ×.

FIG. 4. Paraffin cross sections of the Pronator Quadratus musclesstained with hematoxylin and eosin and examined under light microscope.Left panel—Pronator Quadratus muscle transfected with VEGF-165 plasmid.Right panel—Pronator Quadratus muscle transfected with EPO plasmid. Topleft picture (VEGF-165 ) demonstrates increased number of vessels andinterstitial cells (presumably—endothelial cells), as compared to rightpicture (EPO-control), magnification ×200. Bottom left picture (VEGF-165) demonstrates increased number of vessels, most small arteries andcapillaries, as compare to right picture (EPO-control). Arrows indicateobvious vascular structures, magnification ×6300.

FIG. 5. Paraffin cross sections of the Pronator Quadratus musclesimmunostained for endothelial cell marker—CD31, and examined underconfocal laser scanning microscope LSM 510, magnification ×400. CD31marker visualized with Cy3 (black), nuclei with nucleic acid stains ToPro-3. Muscle fibers and red blood cells were visualized by 488 nm laserhaving autofluorescent emission. Left picture—Pronator Quadratus muscletransfected with VEGF-165 plasmid, demonstrates increased of endothelialcells and small vessels, as compare to right picture (EPO-control). Thenumber of CD31 positive cells was increased significantly in VEGF-165transfected muscle by 61.7% (p<0.001).

DETAILED DESCRIPTION

We have found that an intravascular route of administration allows apolynucleotide to be delivered to parenchymal cells in a more evendistribution than direct parenchymal injections. The efficiency ofpolynucleotide delivery and expression is increased by increasing thepermeability of the tissue's blood vessel. Permeability is increased byone or more of the following: increasing the intravascular pressure,delivering the injection fluid rapidly (injecting the injection fluidrapidly), using a large injection volume, inhibiting vessel fluid flow,and increasing permeability of the vessel wall. Prior to insertion,subsequent to insertion, or concurrent with insertion the permeabilityof the vessel is increased using an exterior cuff thereby the geneticmaterial is delivered to the parenchymal cell.

We describe a process for inserting a polynucleotide into mammaliancells. More particularly we have injected Rhesus macaque monkey limbsand caused the polynucleotide to be delivered and expressed. For boththe arm and leg injections, blood flow was impeded by a cuff surroundingthe arm or leg. The high luciferase and β-galactosidase levels achievedin monkeys indicate that the procedure is likely to be efficient inhumans. It is noteworthy that expression levels were similar in monkeysas those levels in rats since the efficiency of many prior art genetransfer techniques is less in larger animals.

The term cuff means a device for impeding fluid flow through mammalianinternal blood vessels. However, for purposes of the claims, cuff refersspecifically to a device applied exterior to the mammal's skin andtouches the skin in a non-invasive manner. In a preferred embodiment,the cuff is a device that applies external pressure to the mammalianskin and thereby pressure is applied internally to the blood vesselwalls. The vessel walls, in an area underneath the cuff, are forced toconstrict in amount sufficient to impede fluid from flowing at a normalrate. Impeding fluid flow into and out of an area such as a limb,combined with injection of a solution containing polynucleotides, causesvascular pressure and vessel permeability to increase in the area, Thus,the fluid and its contents (including polynucleotides) are urged out ofthe vessel walls and into the extravascular space. One example of a cuffis a sphygmomanometer which is normally used to measure blood pressure.In a preferred embodiment of this specification, the sphygmomanometer isused to apply pressure to mammalian skin, around a limb, for the purposeof increasing vessel permeability when combined with solution injectioninto the vessel. Another example is a tourniquet.

In yet another preferred embodiment the use of a cuff (or other externalpressure device) is combined with the use of a pharmaceutical orbiologically-active agent (such as papaverine) to increase vascularpermeability.

The term intravascular refers to an intravascular route ofadministration that enables a polymer, oligonucleotide, orpolynucleotide to be delivered to cells more evenly distributed thandirect injections. Intravascular herein means within an internal tubularstructure called a vessel that is connected to a tissue or organ withinthe body of an animal, including mammals. Within the cavity of thetubular structure, a bodily fluid flows to or from the body part.Examples of bodily fluid include blood, lymphatic fluid, or bile.Examples of vessels include arteries, arterioles, capillaries, venules,sinusoids, veins, lymphatics, and bile ducts. The intravascular routeincludes delivery through the blood vessels such as an artery or a vein.

Afferent blood vessels of organs are defined as vessels in which bloodflows toward the organ or tissue under normal physiologic conditions.Efferent blood vessels are defined as vessels in which blood flows awayfrom the organ or tissue under normal physiologic conditions. In theheart, afferent vessels are known as coronary arteries, while efferentvessels are referred to as coronary veins.

The term naked nucleic acids indicates that the nucleic acids are notassociated with a transfection reagent or other delivery vehicle that isrequired for the nucleic acid to be delivered to a target cell. Atransfection reagent is a compound or compounds used in the prior artthat mediates nucleic acid's entry into cells.

A polynucleotide may be a nucleic acid that recombines with chromosomalDNA.

Expression cassette refers to a natural or recombinantly producednucleic acid which is capable of expressing protein(s). A DNA expressioncassette typically includes a “promoter” (allowing transcriptioninitiation), and a sequence encoding one or more proteins(“transgene(s)”). Optionally, the expression cassette may includetrancriptional enhancers, locus control regions, matrix attachmentregions, scaffold attachment regions, non-coding sequences, splicingsignals, transcription termination signals, and polyadenylation signals.An RNA expression cassette typically includes a translation initiationcodon (allowing translation initiation), and a sequence encoding one ormore proteins. Optionally, the expression cassette may includetranslation termination signals, a polyadenosine sequence, internalribosome entry sites (IRES), and non-coding sequences.

The expression cassette promoter can be selected from any of the knownpromoters isolated from the group consisting of, but not limited to, thehuman genome, mammalian genomes, microbial genomes, and chimericsequences. Additionally, artificially constructed sequences can be usedthat have shown to have promoter activity in the target cell type.Examples of viral promoters that have successfully been used to expresstransgenes include: human cytomegalovirus immediate early promoter, Roussarcoma virus, Moloney leukemia virus, and SV40. Examples of mammalianpromoters include: elongation factor 1, muscle creatine kinase, actin,desmin, and troponin. The choice of promoter in conjunction with otherexpression cassette elements can determine the level of transgeneprotein production in target cells. The expression cassette can bedesigned to express preferentially in specific cell types (operationallydefined as a 5-fold higher expression level in the specific cell typecompared to the average expression level in other cell types). Apromoter, or combination of a promoter and other regulatory elements inthe expression cassette, resulting in preferential expression inspecific cell types is frequently referred to as tissue-specific. Anexample of a tissue-specific promoter is the muscle creatine kinasepromoter, which expresses transgenes at high levels in skeletal musclecells, whereas expression in other cell types is at lower levels.Preferential expression in muscle cells can be achieved by usingpromoters and regulatory elements from muscle-specific genes (e.g.,muscle creatine kinase, myosin light chain, desmin, skeletal actin), orby combining transcriptional enhancers from muscle-specific genes with apromoter normally active in many cell types (e.g., the humancytomegalovirus immediate early promoter in combination with the myosinlight chain enhancer).

Protein refers herein to a linear series of greater than 2 amino acidresidues connected one to another as in a polypeptide. A therapeuticeffect of the protein in attenuating or preventing the disease state canbe accomplished by the protein either staying within the cell, remainingattached to the cell in the membrane, or being secreted and dissociatedfrom the cell where it can enter the general circulation and blood.Secreted proteins that can be therapeutic include hormones, cytokines,interferons, enzymes (e.g. lysosomal enzymes), growth factors, clottingfactors, anti-protease proteins (e.g., alpha1-antitrypsin), angiogenicproteins (e.g., vascular endothelial growth factor, fibroblast growthfactors), anti-angiogenic proteins (e.g., endostatin, angiostatin), andother proteins that are present in the blood. Proteins on the membranecan have a therapeutic effect by providing a receptor for the cell totake up a protein or lipoprotein (e.g., low density lipoproteinreceptor). Therapeutic proteins that stay within the cell (intracellularproteins) can be enzymes that clear a circulating toxic metabolite as inphenylketonuria. They can also cause a cancer cell to be lessproliferative or cancerous (e.g., less metastatic), or interfere withthe replication of a virus. Intracellular proteins can be part of thecytoskeleton (e.g., actin, dystrophin, myosins, sarcoglycans,dystroglycans) and thus have a therapeutic effect in cardiomyopathiesand musculoskeletal diseases (e.g., Duchenne muscular dystrophy,limb-girdle disease). Other therapeutic proteins of particular interestto treating heart disease include polypeptides affecting cardiaccontractility (e.g., calcium and sodium channels), inhibitors ofrestenosis (e.g., nitric oxide synthetase), angiogenic factors, andanti-angiogenic factors.

Constructs to Improve Secretion of Muscle Expressed Protein into theBlood

Proteins are targeted for secretion from cells by the presence of asignal peptide. During transit through the endoplasmic reticulum, thesignal peptide is removed by specific proteolytic cleavage. It can beanticipated that secretion of certain proteins can be improved byreplacing the endogenous signal peptide with a heterologous signalpeptide. This can be accomplished by exchanging the coding regions forthe signal peptides in the nucleic acid. For example, the signal fromthe protein placental alkaline phosphatase (often used in a truncatedfrom as secreted alkaline phosphatase, SEAP) can be used to replace thesignal from the protein factor IX. This may result in better secretionof the factor IX protein from muscle cells. Since the signal peptide iscleaved prior to secretion, the secreted mature factor IX protein isunaltered and functional. Alternatively, one can construct a fusionbetween the complete SEAP and target protein, or use other definedprotein sequence known to enhance transmembrane transport, such as theTAT protein from the human immunodeficiency virus, or the VP22 proteinfrom herpesviruses.

There are three types of reporter (or marker) gene products that areexpressed from reporter genes. The reporter gene/protein systemsinclude:

Intracellular gene products such as luciferase, β-galactosidase, orchloramphenicol acetyl transferase. Typically, they are enzymes whoseenzymatic activity can be easily measured. Intracellular gene productssuch as β-galactosidase or green fluorescent protein which identifycells expressing the reporter gene. On the basis of the intensity ofcellular staining, these reporter gene products also yield qualitativeinformation concerning the amount of foreign protein produced per cell.

Secreted gene products such as growth hormone, factor IX, secretedalkaline phosphatase, or alpha1-antitrypsin are useful for determiningthe amount of a secreted protein that a gene transfer procedure canproduce. The reporter gene product can be assayed in a small amount ofblood.

We have disclosed gene expression achieved from reporter genes inspecific tissues. The terms therapeutic and therapeutic results aredefined in this application as a nucleic acid which is transfected intoa cell, in vivo, resulting in a gene product (e.g. protein) beingexpressed in the cell or secreted from the cell. Levels of a geneproduct, including reporter (marker) gene products, are measured whichthen indicate a reasonable expectation of similar amounts of geneexpression by transfecting other nucleic acids. Levels of treatmentconsidered beneficial by a person having ordinary skill in the art ofgene therapy differ from disease to disease, for example: Hemophilia Aand B are caused by deficiencies of the X-linked clotting factors VIIIand IX, respectively. Their clinical course is greatly influenced by thepercentage of normal serum levels of factor VIII or IX: <2%, severe;2–5%, moderate; and 5–30% mild. This indicates that in severe patientsan increase from 1% to 2% of the normal level can be consideredbeneficial. Levels greater than 6% prevent spontaneous bleeds but notthose secondary to surgery or injury. A person having ordinary skill inthe art of gene therapy would reasonably anticipate beneficial levels ofexpression of a gene specific for a disease based upon sufficient levelsof marker gene results. In the hemophilia example, if marker genes wereexpressed to yield a protein at a level comparable in volume to 2% ofthe normal level of factor VIII, it can be reasonably expected that thegene coding for factor VIII would also be expressed at similar levels.

Parenchymal Cells

Parenchymal cells are the distinguishing cells of a gland or organcontained in and supported by the connective tissue framework. Theparenchymal cells typically perform a function that is unique to theparticular organ. The term “parenchymal” often excludes cells that arecommon to many organs and tissues such as fibroblasts and endothelialcells within blood vessels.

In a liver organ, the parenchymal cells include hepatocytes, Kupffercells and the epithelial cells that line the biliary tract and bileductules. The major constituent of the liver parenchyma are polyhedralhepatocytes (also known as hepatic cells) that presents at least oneside to an hepatic sinusoid and opposed sides to a bile canaliculus.Liver cells that are not parenchymal cells include cells within theblood vessels such as the endothelial cells or fibroblast cells. In onepreferred embodiment hepatocytes are targeted by injecting thepolynucleotide within the tail vein of a rodent such as a mouse.

In striated muscle, the parenchymal cells include myoblasts, satellitecells, myotubules, and myofibers. In cardiac muscle, the parenchymalcells include the myocardium also known as cardiac muscle fibers orcardiac muscle cells and the cells of the impulse connecting system suchas those that constitute the sinoatrial node, atrioventricular node, andatrioventricular bundle. In one preferred embodiment striated musclesuch as skeletal muscle or cardiac muscle is targeted by injecting thepolynucleotide into the blood vessel supplying the tissue. In skeletalmuscle an artery is the delivery vessel; in cardiac muscle, an artery orvein is used.

Polymers

A polymer is a molecule built up by repetitive bonding together ofsmaller units called monomers. In this application the term polymerincludes both oligomers which have two to about 80 monomers and polymershaving more than 80 monomers. The polymer can be linear, branchednetwork, star, comb, or ladder types of polymer. The polymer can be ahomopolymer in which a single monomer is used or can be copolymer inwhich two or more monomers are used. Types of copolymers includealternating, random, block and graft.

One of our several methods of nucleic acid delivery to cells is the useof nucleic acid-polycations complexes. It was shown that cationicproteins like histones and protamines or synthetic polymers likepolylysine, polyarginine, polyornithine, DEAE dextran, polybrene, andpolyethylenimine are effective intracellular delivery agents while smallpolycations like spermine are ineffective.

A polycation is a polymer containing a net positive charge, for examplepoly-L-lysine hydrobromide. The polycation can contain monomer unitsthat are charge positive, charge neutral, or charge negative, however,the net charge of the polymer must be positive. A polycation also canmean a non-polymeric molecule that contains two or more positivecharges. A polyanion is a polymer containing a net negative charge, forexample polyglutamic acid. The polyanion can contain monomer units thatare charge negative, charge neutral, or charge positive, however, thenet charge on the polymer must be negative. A polyanion can also mean anon-polymeric molecule that contains two or more negative charges. Theterm polyion includes polycation, polyanion, zwitterionic polymers, andneutral polymers that contain equal amounts of anions and cations. Theterm zwitterionic refers to the product (salt) of the reaction betweenan acidic group and a basic group that are part of the same molecule.Salts are ionic compounds that dissociate into cations and anions whendissolved in solution. Salts increase the ionic strength of a solution,and consequently decrease interactions between nucleic acids with othercations.

In one embodiment, polycations are mixed with polynucleotides forintravascular delivery to a cell. Polycations provide the advantage ofallowing attachment of DNA to the target cell surface. The polymer formsa cross-bridge between the polyanionic nucleic acids and the polyanionicsurfaces of the cells. As a result the main mechanism of DNAtranslocation to the intracellular space might be non-specificadsorptive endocytosis which may be more effective then liquidendocytosis or receptor-mediated endocytosis. Furthermore, polycationsare a very convenient linker for attaching specific receptors to DNA andas result, DNA-polycation complexes can be targeted to specific celltypes.

Additionally, polycations protect DNA in complexes against nucleasedegradation. This is important for both extra- and intracellularpreservation of DNA. The endocytic step in the intracellular uptake ofDNA-polycation complexes is suggested by results in which DNA expressionis only obtained by incorporating a mild hypertonic lysis step (eitherglycerol or DMSO). Gene expression is also enabled or increased bypreventing endosome acidification with NH₄Cl or chloroquine.Polyethylenimine which facilitates gene expression without additionaltreatments probably disrupts endosomal function itself. Disruption ofendosomal function has also been accomplished by linking the polycationto endosomal-disruptive agents such as fusion peptides membrane activecompounds, or adenoviruses.

Membrane Active Compounds

Many biologically active compounds, in particular large and/or chargedcompounds, are incapable of crossing biological membranes. In order forthese compounds to enter cells, the cells must either take them up byendocytosis, into endosomes, or there must be a disruption of thecellular membrane to allow the compound to cross. In the case ofendosomal entry, the endosomal membrane must be disrupted to allow forthe entrance of the compound in the interior of the cell. Therefore,either entry pathway into the cell requires a disruption of the cellularmembrane. There exist compounds termed membrane active compounds thatdisrupt membranes. One can imagine that if the membrane active agentwere operative in a certain time and place it would facilitate thetransport of the biologically active compound across the biologicalmembrane. The control of when and where the membrane active compound isactive is crucial to effective transport. If the membrane activecompound is too active or active at the wrong time, then no transportoccurs or transport is associated with cell rupture and thereby celldeath. Nature has evolved various strategies to allow for membranetransport of biologically active compounds including membrane fusion andthe use membrane active compounds whose activity is modulated such thatactivity assists transport without toxicity. Many lipid-based transportformulations rely on membrane fusion and some membrane active peptides'activities are modulated by pH. In particular, viral coat proteins areoften pH-sensitive, inactive at neutral or basic pH and active under theacidic conditions found in the endosome.

Polycations also cause DNA condensation. The volume which one DNAmolecule occupies in complex with polycations is drastically lower thanthe volume of a free DNA molecule. The size of DNA/polymer complex maybe important for gene delivery in vivo. In terms of intravenousinjection, DNA must cross the endothelial barrier and reach theparenchymal cells of interest.

The average diameter of liver fenestrae (holes in the endothelialbarrier) is about 100 nm, and increases in pressure and/or permeabilitycan increase the size of the fenestrae. The fenestrae size in otherorgans is usually less. The size of the DNA complexes is also importantfor the cellular uptake process. After binding to the target cells theDNA-polycation complex is expected to be taken up by endocytosis.

Polymers may incorporate compounds that increase their utility. Thesegroups can be incorporated into monomers prior to polymer formation orattached to the polymer after its formation. The gene transfer enhancingsignal (Signal) is defined in this specification as a molecule thatmodifies the nucleic acid complex and can direct it to a cell location(such as tissue cells) or location in a cell (such as the nucleus)either in culture or in a whole organism. By modifying the cellular ortissue location of the foreign gene, the expression of the foreign genecan be enhanced.

The gene transfer enhancing signal can be a protein, peptide, lipid,steroid, sugar, carbohydrate, nucleic acid or synthetic compound. Thegene transfer enhancing signals enhance cellular binding to receptors,cytoplasmic transport to the nucleus and nuclear entry or release fromendosomes or other intracellular vesicles.

Nuclear localizing signals enhance the targeting of the gene intoproximity of the nucleus and/or its entry into the nucleus. Such nucleartransport signals can be a protein or a peptide such as the SV40 large Tag NLS or the nucleoplasmin NLS. These nuclear localizing signalsinteract with a variety of nuclear transport factors such as the NLSreceptor (karyopherin alpha) which then interacts with karyopherin beta.The nuclear transport proteins themselves could also function as NLS'ssince they are targeted to the nuclear pore and nucleus.

Signals that enhance release from intracellular compartments (releasingsignals) can cause DNA release from intracellular compartments such asendosomes (early and late), lysosomes, phagosomes, vesicle, endoplasmicreticulum, golgi apparatus, trans golgi network (TGN), and sarcoplasmicreticulum. Release includes movement out of an intracellular compartmentinto cytoplasm or into an organelle such as the nucleus. Releasingsignals include chemicals such as chloroquine, bafilomycin or BrefeldinA1 and the ER-retaining signal (KDEL sequence), viral components such asinfluenza virus hemagglutinin subunit HA-2 peptides and other types ofamphipathic peptides.

Cellular receptor signals are any signal that enhances the associationof the gene with a cell. This can be accomplished by either increasingthe binding of the gene to the cell surface and/or its association withan intracellular compartment, for example: ligands that enhanceendocytosis by enhancing binding the cell surface. This includes agentsthat target to the asialoglycoprotein receptor by usingasialoglycoproteins or galactose residues. Other proteins such asinsulin, EGF, or transferrin can be used for targeting. Peptides thatinclude the RGD sequence can be used to target many cells. Chemicalgroups that react with sulfhydryl or disulfide groups on cells can alsobe used to target many types of cells. Folate and other vitamins canalso be used for targeting. Other targeting groups include moleculesthat interact with membranes such as lipids fatty acids, cholesterol,dansyl compounds, and amphotericin derivatives. In addition viralproteins could be used to bind cells.

Polynucleotides

The term polynucleotide, or nucleic acid, is a term of art that refersto a polymer containing at least two nucleotides. Nucleotides are themonomeric units of polynucleotide polymers. Polynucleotides with lessthan 120 monomeric units are often called oligonucleotides. Naturalnucleic acids have a deoxyribose- or ribose-phosphate backbone. Anartificial or synthetic polynucleotide is any polynucleotide that ispolymerized in vitro or in a cell free system and contains the same orsimilar bases but may contain a backbone of a type other than thenatural ribose-phosphate backbone. These backbones include: PNAs(peptide nucleic acids), phosphorothioates, phosphorodiamidates,morpholinos, and other variants of the phosphate backbone of nativenucleic acids. Bases include purines and pyrimidines, which furtherinclude the natural compounds adenine, thymine, guanine, cytosine,uracil, inosine, and natural analogs. Synthetic derivatives of purinesand pyrimidines include, but are not limited to, modifications whichplace new reactive groups such as, but not limited to, amines, alcohols,thiols, carboxylates, and alkylhalides. The term base encompasses any ofthe known base analogs of DNA and RNA including, but not limited to,4-acetylcytosine, 8-hydroxy-N6-methyladenosine, aziridinylcytosine,pseudoisocytosine, 5-(carboxyhydroxylmethyl)uracil, 5-fluorouracil,5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil,5-carboxymethyl-aminomethyluracil, dihydrouracil, inosine,N6-isopentenyladenine, 1-methyladenine, 1-methylpseudo-uracil,1-methylguanine, 1-methylinosine, 2,2-dimethyl-guanine, 2-methyladenine,2-methylguanine, 3-methyl-cytosine, 5-methylcytosine, N6-methyladenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxy-amino-methyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarbonylmethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid methylester,uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil, queosine,2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,5-methyluracil, N-uracil-5-oxyacetic acid methylester,uracil-5-oxyacetic acid, pseudouracil, queosine, 2-thiocytosine, and2,6-diaminopurine. The term polynucleotide includes deoxyribonucleicacid (DNA) and ribonucleic acid (RNA) and combinations on DNA, RNA andother natural and synthetic nucleotides.

DNA may be in form of cDNA, in vitro polymerized DNA, plasmid DNA, partsof a plasmid DNA, genetic material derived from a virus, linear DNA,vectors (P1, PAC, BAC, YAC, artificial chromosomes), expressioncassettes, chimeric sequences, recombinant DNA, chromosomal DNA, anoligonucleotide, anti-sense DNA, or derivatives of these groups. RNA maybe in the form of oligonucleotide RNA, tRNA (transfer RNA), snRNA (smallnuclear RNA), rRNA (ribosomal RNA), mRNA (messenger RNA), in vitropolymerized RNA, recombinant RNA, chimeric sequences, anti-sense RNA,siRNA (small interfering RNA), ribozymes, or derivatives of thesegroups. An anti-sense polynucleotide is a polynucleotide that interfereswith the function of DNA and/or RNA. Antisense polynucleotides include,but are not limited to: morpholinos, 2′-O-methyl polynucleotides, DNA,RNA and the like. SiRNA comprises a double stranded structure typicallycontaining 15–50 base pairs and preferably 21–25 base pairs and having anucleotide sequence identical or nearly identical to an expressed targetgene or RNA within the cell. Interference may result in suppression ofexpression. The polynucleotide can be a sequence whose presence orexpression in a cell alters the expression or function of cellular genesor RNA. In addition, DNA and RNA may be single, double, triple, orquadruple stranded. Double, triple, and quadruple strandedpolynucleotide may contain both RNA and DNA or other combinations ofnatural and/or synthetic nucleic acids.

A polynucleotide can be delivered to a cell to express an exogenousnucleotide sequence, to inhibit, eliminate, augment, or alter expressionof an endogenous nucleotide sequence, or to express a specificphysiological characteristic not naturally associated with the cell.Polynucleotides may be coded to express a whole or partial protein, ormay be anti-sense.

A delivered polynucleotide can stay within the cytoplasm or nucleusapart from the endogenous genetic material. Alternatively, the polymercould recombine (become a part of) the endogenous genetic material. Forexample, DNA can insert into chromosomal DNA by either homologous ornon-homologous recombination.

RNA function inhibitor. A RNA function inhibitor comprises any nucleicacid or nucleic acid analog containing a sequence whose presence orexpression in a cell causes the degradation of or inhibits the functionor translation of a specific cellular RNA, usually a mRNA, in asequence-specific manner. An RNA function inhibitor may also inhibit thetranscription of a gene into RNA. Inhibition of RNA can thus effectivelyinhibit expression of a gene from which the RNA is transcribed. RNAfunction inhibitors are selected from the group comprising: siRNA,interfering RNA or RNAi, dsRNA, RNA Polymerase III transcribed DNAs,ribozymes, and antisense nucleic acid, which may be RNA, DNA, orartificial nucleic acid. SiRNA comprises a double stranded structuretypically containing 15–50 base pairs and preferably 21–25 base pairsand having a nucleotide sequence identical or nearly identical to anexpressed target gene or RNA within the cell. Antisense polynucleotidesinclude, but are not limited to: morpholinos, 2′-O-methylpolynucleotides, DNA, RNA and the like. RNA polymerase III transcribedDNAs contain promoters, such as the U6 promoter. These DNAs can betranscribed to produce small hairpin RNAs in the cell that can functionas siRNA or linear RNAs that can function as antisense RNA. The RNAfunction inhibitor may be polymerized in vitro, recombinant RNA, containchimeric sequences, or derivatives of these groups. The RNA functioninhibitor may contain ribonucleotides, deoxyribonucleotides, syntheticnucleotides, or any suitable combination such that the target RNA and/orgene is inhibited. In addition, these forms of nucleic acid may besingle, double, triple, or quadruple stranded.

Vectors are polynucleic molecules originating from a virus, a plasmid,or the cell of a higher organism into which another nucleic fragment ofappropriate size can be integrated; vectors typically introduce foreignDNA into host cells, where it can be reproduced. Examples are plasmids,cosmids, and yeast artificial chromosomes; vectors are often recombinantmolecules containing DNA sequences from several sources. A vectorincludes a viral vector: for example, adenovirus; DNA; adenoassociatedviral vectors (AAV) which are derived from adenoassociated viruses andare smaller than adenoviruses; and retrovirus (any virus in the familyRetroviridae that has RNA as its nucleic acid and uses the enzymereverse transcriptase to copy its genome into the DNA of the host cell'schromosome; examples include VSV G and retroviruses that containcomponents of lentivirus including HIV type viruses).

A vector is used in this specification to mean any DNA molecule whichcould include associate molecules to transfer DNA sequences into a cellfor expression. Examples include naked DNA, non-viral DNA complexes(e.g. DNA plus polymers [cationic or anionic], DNA plus transfectionenhancing compounds, and DNA plus amphipathic compounds) and viralparticles.

A non-viral vector is defined as a vector that is not assembled withinan eukaryotic cell including non-viral DNA/polymer complexes, DNA withtransfection enhancing compounds and DNA+amphipathic compounds.

Skin is the external covering of a mammalian body including theepidermis, the dermis, and the subcutaneous tissue.

Permeability

In another preferred embodiment, the permeability of the vessel isincreased. Efficiency of polynucleotide delivery and expression wasincreased by increasing the permeability of a blood vessel within thetarget tissue. Permeability is defined here as the propensity formacromolecules such as polynucleotides to move through vessel walls andenter the extravascular space. One measure of permeability is the rateat which macromolecules move through the vessel wall and out of thevessel. Another measure of permeability is the lack of force thatresists the movement of polynucleotides being delivered to leave theintravascular space.

To obstruct, in this specification, is to block or inhibit inflow oroutflow of blood in a vessel. Rapid injection may be combined withobstructing the outflow to increase permeability. For example, anafferent vessel supplying an organ is rapidly injected and the efferentvessel draining the tissue is ligated transiently. The efferent vessel(also called the venous outflow or tract) draining outflow from thetissue is also partially or totally impeded for a period of timesufficient to allow delivery of a polynucleotide. In the reverse, anefferent is injected and an afferent vessel flow is impeded.

In another preferred embodiment, the intravascular pressure of a bloodvessel is increased by increasing the osmotic pressure within the bloodvessel. Typically, hypertonic solutions containing salts such as NaCl,sugars or polyols such as mannitol are used. Hypertonic means that theosmolarity of the injection solution is greater than physiologicosmolarity. Isotonic means that the osmolarity of the injection solutionis the same as the physiological osmolarity (the tonicity or osmoticpressure of the solution is similar to that of blood). Hypertonicsolutions have increased tonicity and osmotic pressure similar to theosmotic pressure of blood and cause cells to shrink.

In another preferred embodiment, the permeability of the blood vesselcan also be increased by a biologically-active molecule. Abiologically-active molecule is a protein or a simple chemical such aspapaverine or histamine that increases the permeability of the vessel bycausing a change in function, activity, or shape of cells within thevessel wall such as the endothelial or smooth muscle cells. Typically,biologically-active molecules interact with a specific receptor orenzyme or protein within the vascular cell to change the vessel'spermeability. Biologically-active molecules include vascularpermeability factor (VPF) which is also known as vascular endothelialgrowth factor (VEGF). Another type of biologically-active molecule canalso increase permeability by changing the extracellular connectivematerial. For example, an enzyme could digest the extracellular materialand increase the number and size of the holes of the connectivematerial. Another type of biologically-active molecule is a chelatorthat binds calcium and thereby increases the endothelium permeability.

In another embodiment a non-viral vector along with a polynucleotide isintravascularly injected in a large injection volume. The injectionvolume is dependent on the size of the animal to be injected and can befrom 1.0 to 3.0 ml or greater for small animals (i.e. tail veininjections into mice). The injection volume for rats can be from 6 to 35ml or greater. The injection volume for primates can be 70 to 200 ml orgreater. The injection volumes in terms of ml/body weight can be 0.03ml/g to 0.1 ml/g or greater.

The injection volume can also be related to the target tissue. Forexample, delivery of a non-viral vector with a polynucleotide to a limbcan be aided by injecting a volume greater than 5 ml per rat limb orgreater than 70 ml for a primate. The injection volumes in terms ofml/limb muscle are usually within the range of 0.6 to 1.8 ml/g of musclebut can be greater. In another example, delivery of a polynucleotide toliver in mice can be aided by injecting the non-viralvector—polynucleotide in an injection volume from 0.6 to 1.8 ml/g ofliver or greater. In another preferred embodiment, delivering apolynucleotide—non-viral vector to a limb of a primate rhesus monkey,the complex can be in an injection volume from 0.6 to 1.8 ml/g of limbmuscle or anywhere within this range.

In another embodiment the injection fluid is injected into a vesselrapidly. The speed of the injection is partially dependent on the volumeto be injected, the size of the vessel to be injected into, and the sizeof the animal. In one embodiment the total injection volume (1–3 mls)can be injected from 5 to 15 seconds into the vascular system of mice.In another embodiment the total injection volume (6–35 mls) can beinjected into the vascular system of rats from 20 to 7 seconds. Inanother embodiment the total injection volume (80–200 mls) can beinjected into the vascular system of monkeys from 120 seconds or less.

In another embodiment a large injection volume is used and the rate ofinjection is varied. Injection rates of less than 0.012 ml per gram(animal weight) per second are used in this embodiment. In anotherembodiment injection rates of less than ml per gram (target tissueweight) per second are used for gene delivery to target organs. Inanother embodiment injection rates of less than 0.06 ml per gram (targettissue weight) per second are used for gene delivery into limb muscleand other muscles of primates.

Cleavable Polymers

A prerequisite for gene expression is that once DNA/polymer complexeshave entered a cell the polynucleotide must be able to dissociate fromthe cationic polymer. This may occur within cytoplasmic vesicles (i.e.endosomes), in the cytoplasm, or the nucleus. We have developed bulkpolymers prepared from disulfide bond containing co-monomers andcationic co-monomers to better facilitate this process. These polymershave been shown to condense polynucleotides, and to release thenucleotides after reduction of the disulfide bond. These polymers can beused to effectively complex with DNA and can also protect DNA fromDNases during intravascular delivery to the liver and other organs.After internalization into the cells the polymers are reduced tomonomers, effectively releasing the DNA, as a result of the strongerreducing conditions (glutathione) found in the cell. Negatively chargedpolymers can be fashioned in a similar manner, allowing the condensednucleic acid particle (DNA+ polycation) to be “recharged” with acleavable anionic polymer resulting in a particle with a net negativecharge that after reduction of disulfide bonds will release thepolynucleic acid. The reduction potential of the disulfide bond in thereducible co-monomer can be adjusted by chemically altering thedisulfide bonds environment. This will allow the construction ofparticles whose release characteristics can be tailored so that thepolynucleic acid is released at the proper point in the deliveryprocess.

pH Cleavable Polymers for Intracellular Compartment Release

A cellular transport step that has importance for gene transfer and drugdelivery is that of release from intracellular compartments such asendosomes (early and late), lysosomes, phagosomes, vesicle, endoplasmicreticulum, Golgi apparatus, trans Golgi network (TGN), and sarcoplasmicreticulum. Release includes movement out of an intracellular compartmentinto cytoplasm or into an organelle such as the nucleus. Chemicals suchas chloroquine, bafilomycin or Brefeldin A1. Chloroquine decreases theacidification of the endosomal and lysosomal compartments but alsoaffects other cellular functions. Brefeldin A, an isoprenoid fungalmetabolite, collapses reversibly the Golgi apparatus into theendoplasmic reticulum and the early endosomal compartment into thetrans-Golgi network (TGN) to form tubules. Bafilomycin A1, a macrolideantibiotic is a more specific inhibitor of endosomal acidification andvacuolar type H⁺-ATPase than chloroquine. The ER-retaining signal (KDELsequence) has been proposed to enhance delivery to the endoplasmicreticulum and prevent delivery to lysosomes.

To increase the stability of DNA particles in serum, we have added topositively-charged DNA-polycation particles polyanions that form a thirdlayer in the DNA complex and make the particle negatively charged. Toassist in the disruption of the DNA complexes, we have synthesizedpolymers that are cleaved in the acid conditions found in the endosome,pH 5–7.

We also have reason to believe that cleavage of polymers in the DNAcomplexes in the endosome assists in endosome disruption and release ofDNA into the cytoplasm.

There are two ways to cleave a polyion: cleavage of the polymer backboneresulting in smaller polyions or cleavage of the link between thepolymer backbone and the ion containing groups resulting in smallionized molecules and a polymer. In either case, the interaction betweenthe polyion and DNA is broken and the number of molecules in theendosome increases. This causes an osomotic shock to the endosomes anddisrupts the endosomes. In the second case, if the polymer backbone ishydrophobic it may interact with the membrane of the endosome. Eithereffect may disrupt the endosome and thereby assist in release of DNA.

To construct cleavable polymers, one may attach the ions or polyionstogether with bonds that are inherently labile such as disulfide bonds,diols, diazo bonds, ester bonds, sulfone bonds, acetals, ketals, enolethers, enol esters, imines, imminiums, and enamines Another approach isconstruct the polymer in such a way as to put reactive groups, i.e.electrophiles and nucleophiles, in close proximity so that reactionbetween the function groups is rapid. Examples include having carboxylicacid derivatives (acids, esters, amides) and alcohols, thiols,carboxylic acids or amines in the same molecule reacting together tomake esters, thiol esters, acid anhydrides or amides.

The present invention additionally provides for the use of polymerscontaining silicon-nitrogen (silazanes) linkages (either in the mainchain of the polymer or in a side chain of the polymer) that aresusceptible to hydrolysis. Hydrolysis of a silazane leads to theformation of a silanol and an amine. Silazanes are inherently moresusceptible to hydrolysis than is the silicon-oxygen-carbon linkage,however, the rate of hydrolysis is increased under acidic conditions.The substitution on both the silicon atom and the amine can affect therate of hydrolysis due to steric and electronic effects. This allows forthe possibility of tuning the rate of hydrolysis of the silizane bychanging the substitution on either the silicon or the amine tofacilitate the desired affect.

In one embodiment, ester acids and amide acids that are labile in acidicenvironments (pH less than 7, greater than 4) to form an alcohol andamine and an anhydride are use in a variety of molecules and polymersthat include peptides, lipids, and multimolecular associations such asliposomes.

In one embodiment, ketals that are labile in acidic environments (pHless than 7, greater than 4) to form a diol and a ketone are use in avariety of molecules and polymers that include peptides, lipids, andliposomes.

In one embodiment, acetals that are labile in acidic environments (pHless than 7, greater than 4) to form a diol and an aldehyde are use in avariety of molecules and polymers that include peptides, lipids, andliposomes.

In one embodiment, enols that are labile in acidic environments (pH lessthan 7, greater than 4) to form a ketone and an alcohol are use in avariety of molecules and polymers that include peptides, lipids, andliposomes.

In one embodiment, iminiums that are labile in acidic environments (pHless than 7, greater than 4) to form an amine and an aldehyde or aketone are use in a variety of molecules and polymers that includepeptides, lipids, and liposomes.

pH-Sensitive Cleavage of Peptides and Polypeptides:

In one embodiment, peptides and polypeptides (both referred to aspeptides) are modified by an anhydride. The amine (lysine), alcohol(serine, threonine, tyrosine), and thiol (cysteine) groups of thepeptides are modified by the an anhydride to produce an amide, ester orthioester acid. In the acidic environment of the internal vesicles (pHless than 6.5, greater than 4.5) (early endosomes, late endosomes, orlysosome) the amide, ester, or thioester is cleaved displaying theoriginal amine, alcohol, or thiol group and the anhydride.

A variety of endosomolytic and amphipathic peptides can be used in thisembodiment. A positively-charged amphipathic/endosomolytic peptide isconverted to a negatively-charged peptide by reaction with theanhydrides to form the amide acids and this compound is then complexedwith a polycation-condensed nucleic acid. After entry into theendosomes, the amide acid is cleaved and the peptide becomes positivelycharged and is no longer complexed with the polycation-condensed nucleicacid and becomes amphipathic and endosomolytic. In one embodiment thepeptides contains tyrosines and lysines. In yet another embodiment, thehydrophobic part of the peptide (after cleavage of the ester acid) is atone end of the peptide and the hydrophilic part (e.g. negatively chargedafter cleavage) is at another end. The hydrophobic part could bemodified with a dimethylmaleic anhydride and the hydrophilic part couldbe modified with a citranconyl anhydride. Since the dimethylmaleyl groupis cleaved more rapidly than the citrconyl group, the hydrophobic partforms first. In another embodiment the hydrophilic part forms alphahelixes or coil-coil structures.

pH-Sensitive Cleavage of Lipids and Liposomes

In another embodiment, the ester, amide or thioester acid is complexedwith lipids and liposomes so that in acidic environments the lipids aremodified and the liposome becomes disrupted, fusogenic or endosomolytic.The lipid diacylglycerol is reacted with an anhydride to form an esteracid. After acidification in an intracellular vesicle the diacylglycerolreforms and is very lipid bilayer disruptive and fusogenic.

Non-Cleavable Polymers

Many cationic polymers such as histone (H1, H2a, H2b, H3, H4, H5), HMGproteins, poly-L-lysine, polyethylenimine, protamine, and poly-histidineare used to compact polynucleic acids to help facilitate gene deliveryin vitro and in vivo. A key for efficient gene delivery using prior artmethods is that the non-cleavable cationic polymers (both in vitro andin vivo) must be present in a charge excess over the DNA so that theoverall net charge of the DNA/polycation complex is positive.Conversely, using our tail vein injection process having non-cleavablecationic polymer/DNA complexes we found that gene expression is mostefficient when the overall net charge of the complexes are negative (DNAnegative charge>polycation positive charge). Tail vein injections usingcationic polymers commonly used for DNA condensation and in vitro genedelivery revealed that high gene expression occurred when the net chargeof the complexes were negative.

Angiogenesis

The term, angiogenesis, in this specification is defined as anyformation of new blood vessels. Angiogenesis may also refer to thesprouting of new blood vessels (endothelium-lined channels such ascapillaries) from pre-existing vessels as a result of proliferation andmigration of endothelial cells. The maturation or enlargement of vesselsvia recruitment of smooth muscle cells, i.e. the formation of collateralarteries from pre-existing arterioles, is termed arteriogenesis.Vasculogenesis refers to the in situ formation of blood vessels fromangioblasts and endothelial precursor cells (EPCs). An anastomosis is aconnection between two blood vessels. The formation of anastomoses canbe important for restoring blood flow to ischemic tissue. The formationof new vessels in ischemic tissue or in other tissue with insufficientblood perfusion is termed revascularization. As used herein, the termangiogenesis encompasses arteriogenesis, vasculogenesis, anastomosisformation, and revascularization.

Angiogenesis is regulated by soluble secreted factors, cell surfacereceptors and transcription factors. Secreted factors include cytokines,chemokines, and growth factors that affect endothelial cells, smoothmuscle cells, monocytes, leukocytes, and precursor cells. Such factorsinclude: vascular endothelial growth factors, fibroblast growth factors,hepatocyte growth factors, angiopoietin 1 (Ang-1), angiopoietin 2(Ang-2), Platelet derived growth factors (PDFGs), granulocytemacrophage-colony stimulating factor, insulin-like growth factor-1(IGF-1), IGF-2, early growth response factor-I (EGR-i), and human tissuekallikrein (HK).

Delivery of genes that encode angiogenic factors to cells in vivoprovides an attractive alternative to repetitive injections of proteinfor the treatment of vascular insufficiency or occlusions. Genes thatencode angiogenic factors, including both natural and recombinantsecreted factors, receptors, and transcription factors, can be targetedto cells in the affected area, thereby limiting deleterious effectsassociated with delivering angiogenic factors throughout the body. Inparticular, according to the described invention, genes for angiogenicfactors can be delivered to muscle cells in vivo, including skeletal andcardiac muscle cells. Expression of the gene and secretion of the geneproduct then induces angiogenesis and improves collateral blood flow inthe targeted tissue. The improved blood flow can both improve muscletissue function and relieve pain associated with vascular diseases.

EXAMPLES

The high luciferase and β-galactosidase levels achieved in monkeysindicate that the procedure is likely to be efficient in humans.Expression levels were somewhat higher in monkeys than in rats.

The intraarterial procedure requires that blood flow be impeded forsubstantially less than the couple of hours of ischemia required fortissue damage. In fact, a common anesthesia for human limb surgery(e.g., carpal tunnel repair) involves the blockage of blood flow forover one hour. We have not observed any widespread histologic evidenceof ischemic muscle damage in rats or primates following the injections.The minimal elevations of muscle-derived enzymes in the serum alsoargues against any consequential muscle damage.

Given that ˜150 ml of fluid is administered to ˜10 kg animals, the largeamount of fluid could adversely affect the animals cardiovascular orhemodynamic status. However, no adverse effects on the animals wereobserved.

The intravascular pressure can be damaging to the arteries. We haveobserved minimal intimal changes in the arteries that are presumed to betransient and without consequence. Nonetheless, this minimal arterialdamage may be prevented by better controlling the intravascularpressure.

For this pDNA administration procedure, several factors limit expressionto the non-target tissue. 1) The tourniquet prevents the immediatespread of vector outside of the limb. 2) efficient pDNA expression inthe non-vascular parenchymal cells requires extravasation of theinjected pDNA.

The procedure requires relatively large amounts of pDNA to beadministered. This has not been associated with any toxic effects inrodents or monkeys. Given that the tourniquet delays pDNA distributionoutside of the limb and the intravascular pDNA is rapidly degraded bycirculating DNases, pDNA toxicity is unlikely. In addition, the cost forproducing clinical grade pDNA is considerably less expensive than viralvectors and does not represent an obstacle to its clinical use.

Example 1

Intraarterial Injections in Monkeys: Seven Rhesus macaque monkeys (5males; 2 females) of 6 to 13.7 kg body weight underwent intraarterialinjections in their limbs following anesthesia with ketamine andhalothane. For the forearm injections, a longitudinal incision, ˜3 cm inlength, was made on the skin along the inside edge of the biceps brachiiand 2 cm above the elbow. After separating the artery from surroundingtissues and veins, a 20 g catheter was inserted into the brachial arteryanterogradely and ligated in place. For the lower leg injections, theprocedure was essentially the same as that used in the arm, but theincision was located on the upper edge of the popliteal fossae and the20 g catheter was inserted into the popliteal artery.

For both the arm and leg injections, blood flow was impeded by asphygmomanometer cuff surrounding the arm or leg proximal to theinjection site. After the sphygmomanometer was inflated to more than 300mmHg air pressure, the catheterized vessels were injected with 30 ml ofnormal saline containing 5 mg papaverine (Sigma Co.). Five min. later, asaline solution containing 100 μg pDNA/ml solution was rapidly injectedwithin 30 to 45 sec. For the arms, the volume of each injection was 75ml and 90 ml in the first two animals and 120 ml thereafter. Theinjection volume was ˜180 ml for the lower legs. The DNA solutions wereinjected using a nitrogen-pressurized cylinder. Two min after injection,the catheters were removed and the sphygmomanometer deflated.

The procedure was initially done on four monkeys in which one arm andleg was injected and muscle biopsies were taken at one (#1–3) or twoweeks (#4). Monkey #2 had to be sacrificed at two weeks after injectionbecause of an eye infection (unrelated to our procedure). Three moremonkeys (#5–7) received an injection in all four extremities (one armand leg on one day and the other two extremities two days later). Musclebiopsies were obtained at one week and the animals were sacrificed attwo weeks after the injections. In monkeys #6 and #7, an arm and legwere injected with pCI-LacZ; all other injections were with pCI-Luc⁺.

Example 2

Reporter Gene Systems: The pCI-Luc⁺ (Promega, Madison, Wis.) andpCI-LacZ plasmids express a cytoplasmic luciferase and the Escherichiacoli LacZ, respectively, from the human cytomegalovirus (CMV)immediate-early promoter. The pCI vector (Promega) also contains an SV40polyadenylation signal. pMI-Luc⁺ was constructed by replacing the CMVpromoter in pCI-Luc⁺ with a 3300-bp murine muscle creatine kinasepromoter. The vector pEBFP-N1 expresses a nuclear-localizing,blue-shifted green fluorescent protein (GFP) from the CMV promoter(Clontech, Palo Alto, Calif.).

Luciferase assays were performed on muscle biopsies, entire muscles andvarious tissues as previously reported. The relative light units (RLU)were converted to nanograms of luciferase by using luciferase standards(Molecular Probes, Eugene, Oreg.) and a standard curve in whichluciferase protein (pg)=RLU×5.1×10⁻⁵.

For the β-galactosidase assays, muscle samples were taken from theproximal, middle, and distal positions of each muscle, cut into smallpieces, frozen in cold isopentane, and stored at −80° C. Muscle pieceswere randomly chosen from each muscle sample (for every position) and 10μm-thick cryostat sections were made. Every tenth section, for a totalof 20 sections, was stained and analyzed. The sections were incubated inX-gal staining solution (5 mM potassium ferricyanide , 5 mM potassiumferrocyanide, 1 mM magnesium chloride, 1 mM X-gal in 0.1 M PBS , pH 7.6)for 4–8 hours at room temperature and counterstained with hematoxylinand eosin. Three sections were selected randomly from the 20 sections ofeach position (usually the 4th, 11th and 17th sections, but an adjacentsection was used if these sections were not intact). As previouslydescribed, the number of β-galactosidase-positive and total cells weredetermined within a cross area in each section by moving the countergrid from the top edge of the section to the bottom and from the leftedge to the right. The percentage of β-galactosidase-positive cells foreach muscle was gotten from the result of positive number divided bytotal cell number. A weighted average for the percent of transfectedcells for each extremity muscle was determined as follows: (ΣAi*Mi)/Mwhere Ai is percent of transfected cells for one muscle, Mi—weight ofthat muscle and M—whole weight of all muscles.

For the co-localization of β-galactosidase and GFP expression, 10μm-thick cryostat sections were fixed with 4% formaldehyde for 5–10 min.Mouse-anti-β-galadosidase antibody and TRITC-labeled goat-anti-mouse IgG(Sigma) were used as primary and secondary antibodies, respectively.Using a Nikon Optiphot epifluorescence microscope with a SenSys CCDCamera (Photometrics, Tucson, Ariz.), two images were collected from thesame view for TRITC-labeled β-galactosidase and for GFP and mergedtogether using the program Adobe Photoshop 4.0.

Example 3

Luciferase and β-galactosidase Expression: Seven rhesus macaque (6–20years old) received pDNA injections into their limb arteries. All sevenmonkeys tolerated the procedure well and had full function of theirarms, hands, legs and feet following the procedure. In particular, thisindicates lack of damage to the radial nerve, which could have beensensitive to the inflated sphygmomanometer surrounding the upper arm.Swelling in the target limbs, a putative correlate of successful genetransfer, was noted afterwards but completely subsided by the next day.When the monkeys awakened from the anesthesia 15 to 30 min after theprocedure, they did not appear to be in any discomfort beyond that ofnormal surgical recovery. Occasionally, the skin in the target limb hadsome spots of hemorrhage that resolved within several days.

Four of the monkeys were sacrificed at 14 to 16 days after injection andall the target muscles of their limbs were assessed for eitherluciferase or β-galactosidase expression (Table 1). These resultsindicate that the intraartery injection of pCI-Luc⁺ DNA yielded levelsof luciferase expression in all muscles of forearm, hand, lower leg andfoot, ranging from 345 to 7332 ng/g muscle (Table 1). The variability inluciferase expression in arm muscles for different animals appearsdependent upon whether the tip of the catheter was positioned in theradial or ulnar artery. The average luciferase expression levels in thelimb muscles were 991.5±187 ng/g for the arm and 1186±673 ng/g for theleg.

After intraarterial injection of pCI-LacZ DNA, β-galactosidaseexpression was found in myofibers. Large numbers ofβ-galactosidase-positive myofibers were found in both leg and armmuscles, ranging from less than 1% to more than 30% in different muscles(Table 1 and FIG. 1). The average percentage for all four limbs injectedwas 7.4%, ranging from 6.3% to 9.9% for each of the limbs. Theβ-galactosidase percentages for specific muscle groups positivelycorrelated with the luciferase levels in the same muscles (r=0.79).

TABLE 1 Mean muscle β-galactosidase or luciferase expression in fourmuscles from monkeys sacrificed two weeks after injection of pCI-LacZ orpCI-Luc⁺. “±” indicates standard error; n indicates the number of limbsassayed. A. Arm muscles β- Luciferase galactosidase (ng/g (% positive)muscle) Muscle group Muscle name (n = 2) (n = 5) Anterior groupSuperficial group palmaris longus 5.9 ± 0.9 2368 ± 1309 pronator teres19.9 ± 9.4  1818 ± 336  flexor carpi radialis 7.8 ± 0.7 1885 ± 762 flexor carpi ulnaris 3.8 ± 3.0 852 ± 314 flexor digitorum spf. 7.7 ± 1.21009 ± 189  Deep group flexor digitorum prof. 1.0 ± 0.5 544 ± 360pronator quadratus 14.3 ± 11.1 1884 ± 331  Posterior group Superficialgroup brachioradialis 9.0 ± 8.7 1148 ± 942  extensor carpi radialis 6.6± 6.3 1179 ± 584  longus extensor carpi radialis 9.4 ± 4.5 1118 ± 325 brevis extensor digitorum 6.2 ± 5.4 1184 ± 94  anconeus 2.0 ± 0.3 1744 ±372  extensor carpi ulnaris 0.6 ± 0.4 371 ± 86  extensor pollicis longus6.9 ± 4.3 927 ± 228 Deep group supinator 15.1 ± 9.3  2398 ± 748 abductor pollicis longus 6.2 ± 3.8 927 ± 228 extensor digiti secund et6.0 ± 5.5 642 ± 168 teriti extensor digiti quart et 4.0 ± 3.5 593 ± 140minimi Muscles of hand muscle of thumb 15.7 ± 0.5  904 ± 494 interosseus17.3 ± 4.3  1974 ± 185  Weighted Average  6.3 ± 0.04 991 ± 187 B. Legmuscles β- Luciferase galactosidase (ng/g (%) muscle) Muscle groupMuscle name (n = 2) (n = 2) Posterior group Superficial groupgastrocnemius 3.0 ± 2.5 743 ± 33  soleus 21.2 ± 1.4  2888 ± 2151 Deepgroup popliteus 37.1 ± 0.5  4423 ± 2657 flexor digitorum longus 8.9 ±2.4 3504 ± 2151 flexor hallucis longus 9.7 ± 2.4 1355 ± 1224 tibialisposterior 28.7 ± 4.3  7332 ± 5117 Anterior group tibialis anterior 2.8 ±0.2 716 ± 162 extensor hallucis longus 4.2 ± 1.4 810 ± 497 extensordigitorum 10.9 ± 1.0  3187 ± 1166 longus abductor hallucis longus 2.2 ±0.2 345 ± 104 Internal group peronaus longus 6.3 ± 2.5 626 ± 383peronaus brevis 8.9 ± 1.3 1300 ± 23  Muscles of foot extensor digitorumbrevis 6.2 ± 5.0 672 ± 607 extensor hallucis brevis 2.4 ± 1.8 672 ± 607LEG MUSCLES Weighted Average 7.3 ± 0.1 1692 ± 768 

Example 4

Toxicity: Serum chemistries and histologic analyses were performed todetermine if the procedure caused any adverse effects in the monkeys.The serum levels of creatine phosphate kinase (CK), alanineaminotransferase, aspartate aminotransferase (AST) and lactatedehydrogenase (LDH) after surgery were several times higher than beforesurgery. Levels peaked at 48 hours post-injection and returning tonormal within several days. Other serum enzymes such asγ-glutamyltransferase (GGT) and alkaline phosphatase, hematologic assays(hematocrit and RBC indices, platelets), serum electrolytes (Na, Cl, K),serum minerals (calcium, phosphate, iron), serum proteins (albumin,total protein), serum lipids (cholesterol, triglycerides), renal indices(urea, creatinine), and bilirubin were unaffected. Total WBC increasedwithin the typical range post-surgery.

Limb muscles were obtained 14 to 16 days after intraarterial injectionand examined histologically. The vast majority of muscle tissue was wellpreserved and did not show any sign of pathology. In a few sections,mononuclear cells were noted surrounding β-galactosidase positivemyofibers, some of which were undergoing degeneration. Immunostainingfor CD-markers indicated that the majority of infiltrating cells wereCD3-positive (T lymphocytes) with only a few B cells.

Example 5

Timecourse of Muscle Expression After Intravascular Injection in RatsMuscle luciferase expression was measured at several time pointsfollowing intravascular delivery of the luciferase gene under control ofeither the CMV promoter (pCI-Luc⁺) or MCK promoter (pMI-Luc⁺) into: a)untreated rats, b) rats continuously immunosuppressed (treated with 2.5mg/kg of FK506 orally and 1 mg/kg dexamethasone subcutaneously one dayprior to, one hour prior to and every day thereafter with FK506) or c)transiently immunosuppressed (treated with 10 mg/kg of FK506 orally and1 mg/kg dexamethasone subcutaneously one day prior to, one hour prior toand one day after intraarterial delivery of pDNA) (Table 2). Inuntreated rats, luciferase expression was lost after 7 days from the CMVpromoter or after 21 days from the MCK promoter. In either pCI-Luc⁺ orpMI-Luc⁺ injected rats, anti-luciferase antibodies were detected usingELISA by day 21 and were present at higher levels at day 56 and 70 afterintravascular pDNA delivery (data not shown).

TABLE 2 Time course of luciferase expression (ng/g muscle) in hindlimbsfollowing intraarterial injections with 500 μg of pCI-Luc+ (A) orpMI-Luc+ (B) into rats treated with various immunosuppression regimens.Time After CONDITION Injection No Transient Continuous (Days) TreatmentImmunnosuppression Immunnosuppression A. pCI-Luc+ 2 990.9 7 492.6 2122.1 30 10.3 672.0 1212.0 56 0.3 70 0.1 17.3 464.0 B. pMI-Luc+ 2 37.3 7499.9 21 286.9 30 1260.0 56 3.3 70 0.3 571.0 1140.0

Example 6

Repetitive Injections: Sprague-Dawley rats (150 g) were injectedintaarterially in the right leg using 500 μg of pCI-Luc⁺ under increasedpressure conditions on day 0. On days 7 and 14 the rats were injectedslowly with 300 μg pCI-Luc⁺ in 1 ml into the tail vein. On day 24, theleft leg was injected intraarterially with 500 μg of pCI-Luc⁺. On day26, the animals were sacrificed and the left leg revealed luciferaseexpression (mean=4,500 ng of total luciferase/leg muscles, n=2) similarto the levels achieved in animals not pre-injected with pDNA (mean=6,940ng/leg muscles, n=26).

In order to explore the ability to access different populations ofmyofibers, the same leg in rats were injected with the 500 μg of theβ-galactosidase vector (pCI-LacZ) and two days hence with 500 μg of thenuclear GFP vector (pEBFP-N1). At two days after the last injection, themuscles were analyzed for expression of the two reporter genes.Expression of GFP and β-galactosidase was most often located indifferent myofibers (FIGS. 2A and C), but in some cells expression wascoincident (FIG. 2B).

Example 7

Labeled pDNA Distribution in Muscle: Rhodamine-labeled pDNA (Rh-pDNA)was injected into the femoral artery of rats under various conditions inorder to explore the uptake mechanism in muscle as was done for liver.When the injections were performed without impeding blood outflow (lowintravascular pressure), almost no DNA was detected within the muscletissues or vessels. FIG. 3A presents a rare field when some DNA can beseen between muscle cells. When the injections were performed withoutflow occlusion (increased intravascular pressure), Rh-pDNA wasdetected throughout all the muscle (FIGS. 3B and C). At 5 min afterinjection, examination of tissue sections indicated that the majority ofthe Rh-pDNA was surrounding the muscle cells and there was nointracellular staining (FIG. 3B, arrow). At one hour after injection,substantial amounts of DNA can be seen inside the cells (FIG. 3C,arrowhead). Examination of serial confocal sections indicates that theintracellular staining pattern is punctate, unlikely consistent with a Ttubular distribution.

Example 8

Expression of a Therapeutic Gene in Skeletal Muscle Tissue: A plasmidDNA (pCI-hF9) expressing the human factor IX gene (cDNA) undertranscriptional control of the human cytomegalovirus promoter wasdelivered to rat hind limb skeletal muscle. A midline abdominal incisionwas made and skin flaps were folded away with clamps to expose targetarea. Intestines were moved to visualize the iliac veins and arteries.Microvessel clips were placed on the external iliac, caudal epigastric,internal iliac, deferent duct, and gluteal arteries and veins to blockboth outflow and inflow of the blood to the leg. An efflux enhancersolution (0.5 mg papaverine in 3 ml saline) was injected into theexternal iliac artery though a 25 g needle, followed by the pDNAcontaining solution (500 μg in 10 ml Ringer's) 5 minutes later. The pDNAsolution was injected in approximately 10 seconds. The microvessel clipswere removed 2 minutes after the injection, and the peritoneum and skinwere closed using sutures. The rats were immunosuppressed by treatmentwith 10 mg/kg of FK506 orally and 1 mg/kg dexamethasone subcutaneouslyone day prior to, one hour prior to, and one day after plasmid DNAdelivery The rats were sacrificed after 3 weeks, at which time the hindlimb skeletal muscles were removed and homogenized in a total volume of60 ml. Human factor IX levels in the rat sera were determined using anELISA and compared to normal human serum. Expression levels in 3 ratswere 1400, 1000, and 1150 ng/ml extract, respectively. Therefore, thetotal amount of human factor IX present in the rat muscle tissue threeweeks after pDNA delivery was approximately 70 μg.

Example 9

Expression of Secreted Alkaline Phosphate from Rat Skeletal MuscleCells: A plasmid DNA expression vector (pMIR54) was constructed in whichthe secreted alkaline phosphatase (SEAP) gene (obtained from plasmidpSEAP-2 basic, Clontech) is under transcriptional control of the humancytomegalovirus promoter. A solution of 500 μg pMIR54 in 10 ml Ringer'swas injected into the iliac artery of Sprague Dawley rats as describedin Examples above. The rats were immunosuppressed by treatment with 2.5mg/kg of FK506 orally and 1 mg/kg dexamethasone subcutaneously one dayprior to, one hour prior to plasmid DNA delivery. Following the pDNAdelivery, the rats were treated with 2.5 mg/kg FK506 daily. Bloodsamples were obtained from these rats at several time points followingplasmid DNA delivery. SEAP expression was determined using achemiluminescent assay (Tropix) and compared to a standard curve.

SEAP expression (ng SEAP per ml serum) Day 7 Day 14 Rat 2889 2,301 1,407Rat 2992 3,735 2,942

Example 10

Expression in Multiple Muscle Groups: 500 μg of pCI-Luc in 10 ml ofnormal saline solution was injected into the femoral artery of adultrats in which a tourniquet was applied to the outside of the legproximal (tourniquet was applied to the upper portion of the quadricepsgroup of muscles) to the injection site. Five days after injection, thedifferent muscle groups from the leg were removed and cut into equalsections. Each section was placed into lysis buffer, the muscles werehomogenized and 10 μl of the resulting lysates were assayed forluciferase activity.

High levels of luciferase expression were expressed in all muscle groupsthat were located distal to the tourniquet. These included the bicepsfemoris, posterior muscles of the upper leg, gastrocnemius, muscles ofthe lower leg, and muscles of the plantar surface.

TABLE 3 Luciferase expression in the various muscles of the rat legafter the injection of 500 μg of pCILuc into the femoral artery with atourniquet applied around the outside of the upper leg muscles. MuscleGroup Total Luciferase (ng/muscle group) Intravascular Delivery to RatLeg (with external tourniquet) Upper leg anterior 0.181* (quadriceps) (*majority of this muscle group was above the tourniquet) Upper leg middle28.3 (biceps femoris) Upper leg posterior 146 (hamstrings) Lower legposterior 253.6 (gastrocnemius) Lower leg anterior 115.2 (lower shinmuscles) Muscles of the plantar 0.433 surface Intravascular Delivery toRat Leg (without tourniquet) Upper leg anterior 0.010 (quadriceps) Upperleg middle 0.011 (biceps femoris) Upper leg posterior 2.16 (hamstrings)Lower leg posterior 1.57 (gastrocnemius) Lower leg anterior 0.72 (lowershin muscles) Muscles of the plantar 0.202 surface

Intravascularly-administered plasmid DNA is expressed efficiently inmultiple muscle groups when blood flow is impeded using an externaltourniquet.

Example 11

Increased Vascularization Following Delivery of a TherapeuticPolynucleotide to Primate Limb

DNA delivery was performed via brachial artery with blood flow blockedby a sphygmomanometer cuff proximately to the injection site. Left armwas transfected with VEGF, while right arm was transfected with EPO. TheSartorious musle from left leg was used as non-injected control. A maleRhesus monkey weighing 14 kg was used for these injections. The animalwas anesthetized with Ketamin (10–15 mg/kg). A modified pediatric bloodpressure cuff was positioned on the upper arm. The brachial artery wascannulated with a 4 F angiography catheter. The catheter was advanced sothat the tip was positioned just below the blood pressure cuff. Prior tothe injection, the blood pressure cuff was inflated so that the cuffpressure was at least 20 mmHg higher than the systolic blood pressure.After cuff inflation, papaverine (5 mg in 30 ml of saline) was injectedby hand (˜8 to 10 seconds). After 5 min, the pDNA solution was deliveredrapidly with a high volume injection system. For the EPO injection, 10mg of pDNA was added to 170 ml of saline and injected at a rate of 6.8ml per second. For the VEGF injection, 10 mg of pDNA was added to 150 mlof saline, and injected at a rate of 5.4 ml per second.

After 65 days, the animal was euthanized by overdose I.V. injection ofpentobarbital Ketamin (10 mg/kg). The entire Pronator Quadratus andPronator teres muscles from both sides were immediately harvested andfixed for 3 day in 10% neutral buffered formalin (VWR, Cleveland, Ohio).After fixation, an identical grossing was performed for left and rightmuscles and slices across the longitudinal muscles were taken. Specimenswere routinely processed and embedded into paraffin (Sherwood Medical,St. Louis, Mo.). Four microns sections were mounted onto precleanedslides, and stained with hematoxylin and eosin (Surgipath, Richmond,Ill.) for pathological evaluation. Sections were examined underAxioplan-2 microscope and pictures were taken with the aid of AxioCamdigital camera (both from Carl Zeiss, Goettingen, Germany).

To evaluate the effect of VEGF plasmid delivery on cell composition inmuscle tissue and neo-angiogenesis, we used monoclonal mouse anti-humanCD31 antibody (DAKO Corporation, Carpinteria Calif.). The immunostainingwas performed using a standard protocol for paraffin sections. Briefly:four microns paraffin sections were deparaffinized and re-hydrated.Antigen retrieval was performed with DAKO Target Retrieval Solution(DAKO Corporation, Carpinteria Calif.) for 20 min at 97° C. To reducenon-specific binding the section were incubated in PBS containing 1%(wt/vol) BSA for 20 min at RT. Primary antibody 1:30 in PBS/BSA wereapplied for 30 min at RT. CD31 antibody were visualized with donkeyanti-mouse Cy3-conjugated IgG, 1:400 (Jackson Immunoresearch Lab, WestGrove Pa.) for 1 h at RT. ToPro-3 (Molecular Probes Inc.) was used fornuclei staining; 1:70,000 dilution incubated for 15 min at RT. Sectionswere mounted with Vectashield non-fluorescent mounting medium andexamined under confocal Zeiss LSM 510 microscope (Carl Zeiss,Goettingen, Germany). Images were collected randomly under 400×magnification, each image representing 0.106 sq mm. Because musclefibers and red blood cells have an autofluorescence in FITC channel weuse 488 nm laser to visualize these structures. Morphometry analysis.Coded mages were opened in Adobe Photoshop 5.5 having image size 7×7inches in 1×7 inches window, and a grid with rulers was overlaid. Thenumber of muscle fibers, CD31 positive cells and total nuclei wascounted in all 7 image's strips consecutively, without any knowledge ofexperimental design. T-Test for Two-Sample Unequal Variances was usedfor statistical analysis.

Results: Microscopic evaluation did not reveal any notable pathology ineither muscle regardless of the gene delivered. Also, neither muscleshowed any notable presence of inflammatory cells, except of fewmacrophages. Necrosis of single muscle fibers was extremely rare inboth, occupying negligible volume and was not associated withinfiltration/vascularization. However, in muscles transfected withVEGF-165 plasmid, the interstitial cell and vascular density (observedin H&E-stained slides) was obviously increased (FIG. 4), as compare toEPO plasmid administered muscle (FIG. 4). Based on morphologicevaluation, these newly arrived interstitial cells we suggested to beendothelial and adventitial cells, smooth muscle cells, and fibroblasts.To evaluate participation of endothelial cells in thisneo-morphogenesis, we have counted the number of CD31 positive cells inEPO and VEGF delivered Pronator quadratus muscles (FIG. 5). To assurethat comparable specimens were analyzed in right and left muscles, thenumber of muscle fibers was counted per area unit (0.106 sq mm). TheVEGF and EPO administered muscles were not different in muscle fibernumber (means 30.5 and 31.6). The number of CD31 positive cells howeverwas significantly increased by 61.7% p<0.001 (means 53.2 vs32.9).

The foregoing is considered as illustrative only of the principles ofthe invention. Furthermore, since numerous modifications and changeswill readily occur to those skilled in the art, it is not desired tolimit the invention to the exact construction and operation shown anddescribed. Therefore, all suitable modifications and equivalents fallwithin the scope of the invention.

1. An in vivo process for improving blood flow in a limb muscle tissue of a mammal comprising: a) inserting an injector into a blood vessel in the limb of the mammal; b) applying a device external to mammalian skin for compressing vessels in an area underneath the device thereby occluding fluid flow into and out of the limb; c) injecting a solution containing polynucleotides encoding a peptide or protein selected from the group consisting of an angiogenic factor and an arteriogenesis factor into the lumen of said blood vessel distal to the occlusion wherein the solution is retained within the limb, thereby increasing the volume of fluid in the limb and delivering the polynucleotides to skeletal muscle cells in the limb; and, d) expressing the polynucleotides in the skeletal muscle cells thereby improving said blood flow in said tissue.
 2. The process of claim 1 wherein the polynucleotides are selected from the group consisting of viral vectors and non-viral vectors.
 3. The process of claim 1 wherein the externally applied device consists of a tourniquet placed over the skin.
 4. The process of claim 1 wherein the externally applied device consists of a cuff placed over the skin.
 5. The process of claim 4 wherein the externally applied device consists of a sphygmomanometer cuff placed over the skin.
 6. The process of claim 1 wherein improving blood flow consists of stimulating new blood vessel formation.
 7. The process of claim 1 wherein the angiogenic factor consists of vascular endothelial growth factor.
 8. The process of claim 1 wherein the angiogenic factor consists of fibroblast growth factor.
 9. The process of claim 8 wherein the fibroblast growth factor is selected from the list consisting of: FGF-1, FGF-1b, FGF-1c, FGF-2, FGF-2b, FGF-2c, FGF-3, FGF-3b, FGF-3c, FGF-4, FGF-5, FGF-7, FGF-9, acidic FGF and basic FGF.
 10. The process of claim 1, wherein expression of the polynucleotides stimulates angiogenesis in the muscle tissue.
 11. The process of claim 1 wherein improving blood flow consists of improving collateral blood flow.
 12. The process of claim 11 wherein improving collateral blood flow consists of stimulating collateral blood vessel formation.
 13. The process of claim 1 wherein the muscle tissue is affected by a vascular occlusion.
 14. The process of claim 1 wherein the muscle tissue is at risk of being affected by a vascular occlusion.
 15. The process of claim 1 wherein the muscle tissue is suffering from ischemia.
 16. The process of claim 1 wherein the muscle tissue is at risk of suffering from ischemia.
 17. The process of claim 1 wherein the mammal has peripheral vascular disease.
 18. The process of claim 1 wherein the mammal has peripheral arterial occlusive disease.
 19. The process of claim 1 wherein the mammal has peripheral-deficient vascular disease.
 20. The process of claim 17 wherein the mammal suffers from claudication or intermittent claudication.
 21. The process of claim 20 wherein delivery of the polynucleotides results in decreased pain associated with a peripheral circulatory disorder.
 22. The process of claim 1 wherein the peptide or protein is secreted from the muscle cell.
 23. The process of claim 1 wherein the peptide or protein stimulates vascular cell growth.
 24. The process of claim 1 wherein delivery of the polynucleotides stimulates vascular cell migration.
 25. The process of claim 1 wherein delivery of the polynucleotides stimulates vascular cell proliferation. 